Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements.

Alcaligenes eutrophus NH9 was isolated from soil. This strain can utilize 3-chlorobenzoate (3-CB) as a sole source of carbon and energy. Most of the 3-CB-negative segregants had lost one of the plasmids present in the parent strain. The genes for catabolism of 3-CB were located within a 9.2-kb SacI fragment of this plasmid (pENH91). The genes were found to hybridize with genes for components of the modified ortho cleavage pathway from Pseudomonas putida. In one of the 3-CB-negative segregants, the plasmid had undergone the deletion of a segment with a size of about 12.5 kb that covered the catabolic genes. The deletion event seemed to be the result of reciprocal recombination between two highly homologous sequences with sizes of 2.5 kb that were present as a direct repeat at the two ends of the region that included the catabolic genes. Nucleotide sequence analysis of homologous fragments revealed a structure that resembled an insertion sequence and relatedness to IS21. During repeated subculturing of NH9 on liquid media with 3-CB, the culture was taken over by a derivative strain (designated NH9A) in which the degradative plasmid carried a duplicate copy of the 12.5-kb region that contained the catabolic genes. The duplication of these genes seemed again to have been mediated by recombination between the direct repeat sequences.     

Development and Application of PCR Primers for the Detection of the tfd Genes in Delftia acidovorans P4a Involved in the Degradation of 2,4‐D

Primers specific for the genes tfdD, tfdE and tfdF, derived from conserved amino acid sequence motifs of the corresponding homologous enzymes, and primers specific for the genes tfdA and tfdB as well as tfdC taken from the literature were applied in PCR reactions using the genomic DNA of Delftia acidovorans P4a as the template. PCR products were obtained with all primer pairs that were similar in size to those found with the genomic DNA of strains harbouring plasmid pJP4 as the carrier of tfd genes. The nucleotide sequences and the corresponding amino acid sequences of the PCR products obtained with Strain P4a were compared with the sequence databases. According to BLAST analyses, the partial sequences of tfdA and tfdB exhibited a 94–99% degree of identity with the homologous sequences of the 2,4‐D‐degrading strains Achromobacter xylosoxidans subsp. denitrificans EST4002 (pEST4011), Burkholderia sp. RASC, Variovorax paradoxus TV1 (pTV1) and Burkholderia cepacia 2a (pIJB1), whereas the partial sequences of the tfdC, tfdD, tfdE and tfdF genes revealed a 96–100% degree of identity with the homologous sequences of the chlorobenzene‐utilizing strains Ralstonia eutropha NH9 (pENH91), Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 (pP51).     

Transposases are responsible for the target specificity of IS1397 and ISKpn1 for two different types of palindromic units (PUs)

Insertion sequences (IS)1397 and ISKpn1, found in Escherichia coli and Klebsiella pneumoniae, respectively, are IS3 family members that insert specifically into short palindromic repeated sequences (palindromic units or PUs). In this paper, we first show that although PUs are naturally absent from extrachromosomal elements, both ISs are able to transpose from the chromosome or from a plasmid into PUs artificially introduced into target plasmids. We also show that ISKpn1 target specificity is restricted to K.pneumoniae Z¹ PU type, whereas IS1397 target specificity is less stringent since the IS targets the three E.coli Y, Z¹ and Z² PU types indifferently. Experiments of transposition of both ISs driven by both transposases demonstrate that the inverted repeats flanking the ISs are not responsible for this target specificity, which is entirely due to the transposase itself. Implications on ISs evolution are presented.     

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.     

Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51.

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.     

ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.     

Genetic organization and transposition properties of IS511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISRI, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5′ end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G?+?C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.     

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10^?8/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10^?8/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.     

Suicide Plasmid-Dependent IS1-Element Untargeted Integration into Aeromonas veronii bv. sobria Generates Brown Pigment-Producing and Spontaneous Pelleting Mutant

Foodborne Gram-negative pathogens belonging to the genus Aeromonas are variable in harboring insertion sequence (IS) elements that play an important role in the generation of dysfunctional relatives of known genes. Using suicide plasmids carrying an IS1-element, untargeted integration is a common problem during experimental trials to generate specific mutations by homologous recombination. In this work, different strains of Aeromonas veronii bv. sobria (AeG1 and ATCC 9071T), A. hydrophila ATCC 19570, and A. sobria ATCC 43979T are examined for acquisition of IS1-element from pYAK1 suicide plasmid. It was found that untargeted integration of IS1-element is encountered only in ATCC 9071T strain. Such untargeted integration generates a novel brown pigment-producing and spontaneous pelleting (BP⁺SP⁺) mutant. Furthermore, BP⁺SP⁺ mutant strain secretes significantly higher quantity of PilF homologous protein than the wild-type strain and displays an enhanced protein tyrosine phosphorylation activity. Thus, current work shows that Aeromonas spp. strains are variable in their susceptibility for suicide plasmid-dependent IS1-element untargeted integration as well as the susceptible strain is changed to mimic pigment-producing and spontaneous pelleting strains that are naturally occurring among heterogeneous group of foodborne aeromonads.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3′-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)₂, similar to the transposition intermediates described for a number of IS elements.     

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements

Summary A revised physical map of the -haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.     

Symbiosis Island Shuffling with Abundant Insertion Sequences in the Genomes of Extra-Slow-Growing Strains of Soybean Bradyrhizobia

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.     

Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803.

The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.     

Characterization and distribution of two insertion sequences,IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encoded by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS 1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981 s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during co-cultures used in the manufacture of cheese.     

ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?

“Common regions” (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5′ sequences via misreading of the cognate terIS, i.e., “unchecked transposition.” Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-β-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via “unchecked RC transposition,” as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their “genetic construction kit” to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.     

Wide-Range Distribution of Insertion Sequences Identified in B. halodurans among Bacilli and a New Transposon Disseminated in Alkaliphilic and Thermophilic Bacilli

All of the insertion sequences (ISs) except for IS663 and agroup II intron identified in the alkaliphilic Bacillus haloduransC-125 genome were also detected in nine other strains of thesame species by PCR and Southern blot analysis. The transposaseof IS653 identified in the genomes of the 10 strains of B. haloduranswas found to have become the most diversified of all ISs identifiedin the genomes of 10 strains. A new IS element designated IS661belonging to the IS1380 family with inverted repeats (IRs) 17bp in length was present within IS658 identified in the genomeof B. halodurans A59. In addition, a new transposon designatedTn3271bh was identified within the IS642 element in the A59genome, which is similar to a transposon identified in thermophilicGeobacillus stearothermophilus T-6. The new transposon, Tn3271bh,generated an 8-bp duplication of the target site sequence andcarries a 21-bp IR. On the other hand, all kinds of ISs exceptfor IS643 and IS658 were distributed in the genome of obligatelyalkaliphilic Bacillus alcalophilus. Three ISs (IS652, IS653,and IS660) and a group II intron (Bh.Int) were widely dispersedin other Bacillus species without a correlation with the phylogeneticplacement based on 16S rDNA sequences.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.